Application of photoactivatable fluorescent active-site directed probes to serine-containing enzymes.
A photoactivatable fluorescent anthraniloyl group has been directed to the active-site serine group of alpha-chymotrypsin and trypsin. The acylated derivatives are nonfluorescent until irradiated. When activated by light a highly reactive nitrene is generated which is capable of covalent insertion into the protein matrix. The resultant insertion product of this photolysis is a highly fluorescent reporter group which has little rotational mobility and is cross-linked through the serine to the protein matrix in the active site region of the protein. Because of the sensitivity to the polarity of the environment shown by the anthraniloyl chromophore, the dipolar relaxation characteristics of the cross-linked through the serine to the protein matrix in the active site region of the protein. Because of the sensitivity to the polarity of the environment shown by the anthraniloyl chromophore, the dipolar relaxation characteristics of the cross-linked enzyme and deacylated enzyme were determined. These measurements show that little relaxation occurs on the nanosecond time scale for the cross-linked enzyme, but upon deacylation of the serine increased dipolar relaxation of the protein with the attached reporter group is observed. The use of these active-site directed photoactivatable fluorescent probes can be extended to probe the active-site structure of complex enzymes and conformational dynamics of active-site regions in proteins and to serve as potential functional site labels in fluorescence resonance energy transfer measurements.
D000345:Affinity Labels / Q000138:chemical synthesis; D001386:Azides / Q000138:chemical synthesis; D001665:Binding Sites; D002918:Chymotrypsin; D005456:Fluorescent Dyes / Q000138:chemical synthesis; D007700:Kinetics; D010782:Photolysis; D011487:Protein Conformation; D012694:Serine / Q000032:analysis*; D013050:Spectrometry, Fluorescence; D013056:Spectrophotometry, Ultraviolet; D014357:Trypsin
D000345:Affinity Labels; D001386:Azides; D005456:Fluorescent Dyes; D012694:Serine; C032148:4-nitrophenyl 2-azidobenzoate; D002918:Chymotrypsin; D014357:Trypsin